Nerve-dependent regulation of H3K27me3 during the induction of patterning competency in regenerating axolotl limb cells

Limb regeneration in the Mexican axolotl requires the dedifferentiation of mature limb cells into progenitor cells known as blastema cells. Blastema cells become responsive to the signals that define the blueprint, or pattern, of the regenerating cells organizing into complex limb structures. Signaling downstream of the limb nerves plays a significant role in inducing patterning competency; however, the foundations of this process remain poorly understood. Studying patterning competency in amputated axolotl limbs is challenging because it occurs within the context of an intricate network of signals and tissue interactions that are activated simultaneously during regeneration. Here, we develop a simplified limb injury model assay called the competency accessory limb model (CALM), which we used as a platform to characterize the specific timing of competency induction and maintenance and identify the H3K27me3 chromatin signatures associated with this cellular state. We also discovered that..., Tissues were harvested, fixed overnight in 4% formaldehyde, decalcified in 10% EDTA (Amresco) for 5 days on an orbital shaker, and then moved into 30% sucrose (Fisher Scientific) for 3 days on an orbital shaker at 4 °C. Samples were then flash frozen in OCT (Sakura) and cut into 10 μm longitudinal tissue sections for immunodetection. After sectioning, the samples were warmed and then washed twice in PBS and once in 1X PBS-Tween to remove OCT. Samples for H3K27me3 detection were then pressure cooked for 2 minutes in sodium citrate buffer. Samples are then washed with diH2O. After blocking with 0.5% BSA, the samples were incubated overnight in a 1:300 solution of rabbit anti-histone H3K27me3 polyclonal antibody (A4039050, Epigentek) in PBS-Tween at 4 °C. Slides were washed and blocked for an additional 10 minutes before being incubated for 2 hours at 4 °C in a 1:200 dilution of goat anti-rabbit IgG H&L secondary antibody (ab150080, Abcam). Samples were then stained with DAPI in PBS-Tw..., , # Data from: Nerve-dependent regulation of H3K27me3 during the induction of patterning competency in regenerating axolotl limb cells

[https://doi.org/10.5061/dryad.tqjq2bw8x](https://doi.org/10.5061/dryad.tqjq2bw8x)

## Description of the data and file structure

This data set supports Supplemental Figure 3 of the publication *Nerve-dependent regulation of H3K27me3 during the induction of patterning competency in regenerating axolotl limb cells*. The images of the compressed folder are the selected cell nuclei images that were stained with DAPI and H3K27me3 (Epigentek A4039050), were capture as Z-stacks using Axio Observer Z1 inverted fluorescence microscope equipped with an EXCELITAS Technologies X-Cite 120 LED lamp, a 40X oil immersion objective with a Hamamatsu digital camera C11440 and an Apotome2 unit. The imaging was controlled by the Zen Pro 2.0 software package. Z-stacks were imaged using 0.24 μm intervals. Raw z-stacks were then converted into apotome calculation stacks using ...